Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.
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PMID:Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues. 130 81

Pig kidney medium-chain acyl-CoA dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human medium-chain acyl-CoA dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation.
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PMID:Reductive half-reaction in medium-chain acyl-CoA dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue. 139 Jun 38

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method is demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.
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PMID:A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS. 139 Sep 41

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

The mitochondrial metabolism of 5-enoyl-CoAs, which are formed during the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of beta-oxidation. Metabolites were identified spectrophotometrically and by high performance liquid chromatography. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis-octadienoyl-CoA, which was isomerized to 3,5-octadienoyl-CoA either by mitochondrial delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) or by peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA in the presence of soluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a delta 3,5,delta 2,4-dienoyl-CoA isomerase. Qualitatively similar results were obtained with 2-trans-5-trans-octadienoyl-CoA formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans-4-trans-Octadienoyl-CoA was a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble extract of rat liver mitochondria catalyzed the isomerization of 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA, which upon addition of NADPH, NAD+, and CoA was chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetyl-CoA. Thus we conclude that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the beta-oxidation of polyunsaturated fatty acids.
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PMID:NADPH-dependent beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms. 149 56

Resonance Raman (RR) spectra of the complex of pig kidney medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA and of the purple complex formed upon the addition of octanoyl-CoA to the dehydrogenase were obtained. RR spectra were also measured for the complexes prepared by using isotopically labeled compounds, i.e., [3-13C]-, [1,3-13C]-, and [2,4-13C2]acetoacetyl-CoA; [1-13C]octanoyl-CoA; the dehydrogenase reconstituted with [4a-13C]- and [4,10a-13C2]FAD. Both bands of oxidized flavin and acetoacetyl-CoA were resonance-enhanced in the 632.8 nm excited spectra of the acetoacetyl-CoA complex; this confirms that the broad long-wavelength absorption band is a charge-transfer absorption band between oxidized flavin and acetoacetyl-CoA. The 1,622 cm-1 band was assigned to the C(3)=O stretching mode coupling with the C(2)-H bending mode of the enolate form of acetoacetyl-CoA and the bands at 1,483 and 1,119 cm-1 were assigned to bands associated with the C(2)=C(1)-O- moiety. Both bands of fully reduced flavin and the substrate were resonance-enhanced in the 632.8 nm excited spectra of the purple complex. As the enzyme is already reduced, the substrate must be oxidized to octenoyl-CoA; the complex is a charge-transfer complex between the reduced enzyme and octenoyl-CoA. The low frequency value of the 1,577 cm-1 band, which is associated with the C(2)-C(1)=O moiety of the octenoyl-CoA, suggests that the enzyme-bound octenoyl-CoA has an appreciable contribution of C(2)=C(1)-O-.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Resonance Raman study on complexes of medium-chain acyl-CoA dehydrogenase. 150 Apr 13

The most prominent biochemical consequence of riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoA dehydrogenase (IVD). As a result, oxidation of fatty acids and leucine is severely inhibited. We studied the effects of FAD at various stages of acyl-CoA dehydrogenase biogenesis. Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. The decreases in IVD and short chain acyl-CoA dehydrogenase were particularly severe, reaching values of 17 and 34% of controls, respectively. With the exception of IVD, the rate of in vitro transcription of the respective genes and the amounts of mRNAs of these flavoproteins in tissues increased 3-8.5-fold over controls. The amount of IVD mRNA and its transcription rate remained unchanged, suggesting that IVD expression is regulated separately from other acyl-CoA dehydrogenases. When riboflavin was depleted, in vitro translation of acyl-CoA dehydrogenase and electron transfer flavoprotein alpha-subunit mRNAs was moderately inhibited. Translation of non-flavoproteins was also inhibited. The stability of precursor acyl-CoA dehydrogenases and their mitochondrial import/processing were unaffected. However, mature acyl-CoA dehydrogenases degraded markedly faster in deficient mitochondria than in controls. Regardless of whether precursors were translated under riboflavin-depleted or riboflavin replete conditions, mature acyl-CoA dehydrogenases survived well when imported into normal mitochondria but degraded faster when imported into deficient mitochondria. These findings indicate that FAD ligand binds to mature acyl-CoA dehydrogenase inside the mitochondria.
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PMID:FAD-dependent regulation of transcription, translation, post-translational processing, and post-processing stability of various mitochondrial acyl-CoA dehydrogenases and of electron transfer flavoprotein and the site of holoenzyme formation. 151 28

Fibroblasts from patients with long-chain acyl-CoA dehydrogenase deficiency were found to oxidize [1-14C]linoleate at an average rate of 60% of normal but [9,10(n)-3H]myristate at an average rate of only 37% of normal, a relationship reverse from that predicted by the chain-length specificities of the three known straight-chain mitochondrial acyl-CoA dehydrogenases. The residual long-chain beta-oxidative activity was found to be mitochondrial and associated with the accumulation of tetradecadienoate (C14:2w6) when the mutant fibroblasts were incubated with 100 mumol/L linoleate (C18:2w6) or eicosadienoate (C20:2w6). The results suggest the presence in human fibroblasts of a novel acyl-CoA dehydrogenase with activity toward 15 to 20 carbon-length fatty acids.
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PMID:Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase. 154 Jan 49

beta-Oxidation of palmitate and tetradecanedioic acid was studied in cell-free extracts of the Gram-positive bacterium Corynebacterium sp. strain 7E1C, and the acyl-CoA ester intermediates formed were analysed by h.p.l.c. beta-Oxidation assays displayed a lag phase before a constant rate of NAD+ reduction was obtained. The length of the lag phase was inversely proportional to the number of units of activity added to assays. This is a characteristic feature of a system of consecutive reactions proceeding via free intermediates. During beta-oxidation of palmitate all the saturated acyl-CoAs from C16 to C8 were detected together with trace amounts of unsaturated and 3-hydroxy-intermediates. The time-course of intermediate formation again indicated a precursor-product relationship indicative of free intermediates being formed. When 3-hydroxyacyl-CoA dehydrogenase was inhibited by completely removing NAD+ from assays, the major acyl-CoAs, detected during palmitate beta-oxidation were palmitoyl-CoA, hexadeca-2-enoyl-CoA and 3-hydroxypalmitoyl-CoA. These compounds also displayed a precursor-product relationship. Under normal assay conditions the acyl-CoA dehydrogenase(s) are the probable rate-limiting enzyme(s) of the beta-oxidation spiral. These results indicate that in cell-free extracts of Corynebacterium sp. strain 7E1C, beta-oxidation proceeds via free acyl-CoA intermediates and is at variance with the concept of substrate channelling or of a 'leaky hose pipe' model as proposed for mitochondrial beta-oxidation in eukaryotic cells. The significant accumulation of chain-shortened acyl-CoA esters is similar to the situation observed for mammalian peroxisomal beta-oxidation.
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PMID:Long-chain acyl-CoA ester intermediates of beta-oxidation of mono- and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C. 163 89

The theory of steady-state flux control was applied to characterize the regulation of beta-oxidation flux in uncoupled rat liver mitochondria oxidizing palmitoylcarnitine in the presence of rotenone, malonate and the beta-hydroxybutyrate/acetoacetate redox buffer. By titrations with inhibitors such as antimycin, myxothiazol, azide and 4-pentenoic acid, the flux control coefficients of the b-c1 complex, cytochrome c oxidase and thiolase, were determined experimentally. The flux control coefficients of carnitine palmitoyltransferase II, ETF:CoQ oxidoreductase and beta-hydroxybutyrate dehydrogenase were determined from elasticity coefficients obtained by measuring the flux dependencies of acyl-CoA and acetyl-CoA+CoASH concentrations, the electron transfer flavoprotein redox state, the CoQ redox state and the NAD redox state. It was found that at low flux rates the flux control was distributed mainly between acyl-CoA dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase (Ci = 0.89). At maximum flux rates, carnitine palmitoyltransferase II (Ci = 0.35) and thiolase (Ci = 0.13) contribute additionally to the flux control. Thus, the phenomena of regulation of mitochondrial beta-oxidation can be described as multistep control.
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PMID:Application of the theory of steady-state flux control to mitochondrial beta-oxidation. 166 35


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